Heavy water induces bundling in entangled actin networks

Heavy water is known to affect many different biological systems, with the most striking effects observed at the cellular level. Many dynamic processes, such as migration or invasion, but also central processes of cell proliferation are measurably inhibited by the presence of deuterium oxide (D2O). Furthermore, individual cell deformabilities are significantly decreased upon D2O treatment. In order to understand the origin of these effects, we studied entangled filamentous actin networks, a commonly used model system for the cytoskeleton, which is considered a central functional element for dynamic cellular processes. Using bulk shear rheology to extract rheological signatures of reconstituted actin networks at varying concentrations of D2O, we found a non-monotonic behavior, which is explainable by a drastic change in the actin network architecture. Applying light scattering and fluorescence microscopy, we were able to demonstrate that the presence of deuterium oxide induces bundling in reconstituted entangled networks of filamentous actin. This constitutes an entirely novel and previously undescribed actin bundling mechanism.


B. Rheology
Shear rheology measurements were performed with a dynamic shear rheometer (ARES, TA Instruments, USA or MCR 502, Anton Paar, Germany) and a cone-plate geometry with a diameter of 25 mm and a gap width of 50 µm. Actin was polymerized between plate and cone for 2 hours at 20 • C after initiating polymerization by adding 10-fold F-buffer and water to a final volume of 175µl. To prevent interfacial elasticity artifacts, the cone was surrounded with a DMPC solution dissolved in dichloromethane at a concentration of 0.3 µg/ml. Following the application, DMPC assembles into a monolayer surrounding the geometry, thereby eliminating air exposure of the polymer solution. The sample chamber was sealed with a cap equipped with wet sponges to prevent evaporation. The measurement sequence consisted of following measurements (i-v): (i) Polymerization was monitored with a dynamic time sweep with one measurement point every 120 s at a frequency of f = 1 Hz and a strain of γ = 5 %. Only those samples that were in equilibrium at the end of the time sweep were considered for further analysis. The linear regime was first measured with (ii) a short dynamic frequency sweep with a strain of γ = 5 % in the range of f = 0.01−10 Hz with 7 points per decade, followed by (iii) a long dynamic frequency sweep with a strain of γ = 5 % in the range of f = 0.001 − 30 Hz with 21 points per decade, before repeating the (iv) short dynamic frequency sweep again (γ = 5 %, f = 0.1 − 10 Hz, 7 points per decade). Lastly, (v) a transient step rate test at a strain rate of 0.1 s −1 was used to measure the non-linear strain regime. The differential shear modulus K was determined from the resulting stress-strain curves with a self-written Python script. K was calculated as the gradient of the spline fit smoothed stress data divided by the strain step width. The linear value K lin was defined at the first non-negative stress value. Negative stress values, particularly for small strains, appear due to measurement limitations as well as a result of the fitting routine.

C. Static light scattering
Static light scattering (Malvern Instruments Ltd., Zetasizer Nano ZSP, UK) at a wavelength of 633 nm was used to observe the dependence of actin network morphology on D 2 O concentrations. The actin concentration was 12 µM . The D 2 O concentration was increased in 10 % increments from 0 % to 70 %. The scattering of the sample was measured every 15 seconds for 0.5 h before scattering intensities were arithmetically averaged.
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D. Spinning disk confocal microscopy
For visualization, monomeric actin was mixed at a molar ratio of 1:1 with phalloidin tetramethylrhodamine isothiocyanate (phalloidin-TRITC) purchased from Sigma-Aldrich. D 2 O was added to yield volume concentrations between 0 % D 2 O and 70 % D 2 O, in increments of 10 %. After mixing all components, polymerization was initialized by adjusting the salt concentration to match 1x F-buffer conditions, with a final actin concentration of 0.04 mg/ml. Immediately after the polymerization process was started, the premixed solution was deposited into a sample chamber as described previously. Measurements were performed on a spinning disc confocal microscope (inverted Axio Observer.Z1/Yokogawa CSU-X1A 5000 (Carl Zeiss Microscopy GmbH, Germany), 100x oil immersion objective (Plan-Apochromat 100x/1.40 Oil DIC M27)) and recorded with a Hama-matsu camera at an exposure time of 100 ms. Fig. 1 shows microscopy images at a D 2 O concentration of 50 %.
FIG. 2. Differential shear modulus normalized by its value in the linear regime K/K lin as a function of measured stress. Based on the same data set as the curves shown in Figure 1d of the main manuscript, this representation allows for determination of the stress values at which networks yield (listed in Table 1). The values of yield stress do not directly correlate to the amount of heavy water present in the solution and exhibit non-monotonic behavior similar to other rheological parameters.